This project is to refine understanding about mechanisms by which the autotoxic catecholaldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL) is built up in affected catecholaminergic neurons in the autonomic synucleinopathies Parkinsons disease (PD), pure autonomic failure (PAF), and multiple system atrophy (MSA) and about mechanisms of DOPAL-induced cytotoxicity in catecholaminergic neurons. We obtained further post-mortem neurochemical evidence in PD and MSA for a double hit of decreased vesicular sequestration and decreased DOPAL detoxification by aldehyde dehydrogenase (ALDH), both abnormalities promoting DOPAL accumulation. DOPAL toxicity involves several mechanisms that come under the broad headings of oxidative stress and protein modifications. The latter seem to depend importantly on spontaneous oxidation of DOPAL. Of special interest is understanding better how DOPAL interacts with alpha-synuclein, since PD, PAF, and MSA all involve abnormal deposits of this protein. In addition, we are pursuing our finding that DOPAL can be transmitted to and aggregate alpha-synuclein in glial cells. To address these matters we are using novel neurochemical methodology to assess simultaneously the spontaneous and enzymatic oxidation of cytoplasmic dopamine, by measuring 5-S-cysteinyldopamine (Cys-DA), DOPAL, and 3,4-dihydroxyphenylacetic acid (DOPAL) simultaneously in cells and tissues; by assaying DOPAL-quinone for the first time; and by visualizing DOPAL-induced synuclein aggregation by immunofluorescence microscopy. (1) Determinants of denervation-independent depletion of putamen dopamine in Parkinson's disease and multiple system atrophy: The severity of putamen dopamine depletion in parkinsonian synucleinopathies exceeds that explained by denervation alone. Decreased activities of tyrosine hydroxylase (TH) or L-aromatic-amino-acid decarboxylase (LAAAD) or decreased vesicular storage in residual terminals could make up the difference. Cysteinyl-DOPA (Cys-DOPA) and cysteinyl-dopamine (Cys-DA) are formed from spontaneous oxidation of the parent compounds in the neuronal cytoplasm. We assayed levels of endogenous catechols including dopamine, its cytoplasmic metabolites (Cys-DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylethanol (DOPET), DOPAL, and 2 tyrosine hydroxylation products proximate to dopamine (DOPA and Cys-DOPA) in post-mortem putamen tissue samples from PD and MSA patients and control subjects. In the synucleinopathy patients an index of vesicular storage of dopamine (the ratio of dopamine to the sum of its cytoplasmic metabolites) averaged 54% of control (p=0.001), an index of L-aromatic-amino-acid decarboxylase (LAAAD) activity averaged 21% of control (p<0.0001), and an index of innervation (the sum of DOPA+Cys-DOPA) averaged 63% of control (p=0.01). Thus, putamen dopamine depletion in these diseases seems to reflect not only nigrostriatal denervation but also functional abnormalities in the residual terminals. These abnormalities include the double hit of decreased vesicular storage and decreased activity of aldehyde dehydrogenase.1 (2) DOPAL is transmissible to and oligomerizes alpha-synuclein in human glial cells: Glial cytoplasmic inclusions (GCIs) containing alpha-synuclein are a neuropathologic hallmark of MSA. Oligomerized alpha-synuclein is thought to be the pathogenic form of the protein. Glial cells normally express little alpha-synuclein, but they can take up alpha-synuclein from the extracellular fluid. DOPAL, an obligate intermediate in the intra-neuronal metabolism of dopamine, potently oligomerizes alpha-synuclein. In this study we tested whether DOPAL is taken up by human glial cells and augments intracellular oligomerization of alpha-synuclein. DOPAL (exogenous or endogenous from co-incubation with PC12 cells) and alpha-synuclein (native or A53T mutant form) were added to the incubation medium of glial cells (glioblastoma or MO3.13 oligodendrocytes). Glial cellular contents of DOPAL and its intracellular metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at up to 180 min of incubation. Neither glioblastoma nor MO3.13 cells contained endogenous catecholamines or alpha-synuclein. Co-incubation of the cells with dopamine-producing PC12 cells produced time-related increases in DOPAL and DOPAC contents. Similarly, glial cellular DOPAL and DOPAC contents increased rapidly after addition of DOPAL to the medium. After addition of native or A53T-alpha-synuclein, intracellular alpha-synuclein also increased. Incubation of glial cells with both DOPAL and alpha-synucleinenhanced the intracellular oligomerization of native and A53T-alpha-synuclein. Therefore, DOPAL is transmissible to glial cells and enhances intracellular oligomerization of AS. An interaction of DOPAL with alpha-synuclein might help explain the formation of CGIs in MSA.2 (3) Immunofluorescence confocal microscopy to visualize sympathetic noradrenergic nerves and alpha-synuclein in PD and related disorders: Within our Section we have begun to visualize tyrosine hydroxylase (TH) and alpha-synuclein simultaneously by immunofluorescence microscopy. We have identified co-localized TH and alpha-synuclein in sympathetic ganglionic neurons in incidental Lewy body disease.3 Preliminarily, applying this technology has successfully visualized DOPAL-induced alpha-synuclein aggregation in human glial cells incubated with DOPAL and alpha-synuclein. (4) Mechanisms of DOPAL toxicity: Although it is known that PD involves putamen buildup of the autotoxic dopamine metabolite, DOPAL, mechanisms of DOPAL cytotoxicity are incompletely understood. We explored oxidative stress, with consequent mitochondrial damage and apoptosis, and protein modifications, including conversion of alpha-synuclein to its toxic oligomeric form and formation of DOPAL-quinone-related protein adducts. Markers of oxidative stress, mitochondrial potential, apoptosis, and synuclein oligomerization were measured during incubation of human MO3.13 oligodentrocytes or rat pheochromocytoma PC12 cells with DOPAL. Effects of the anti-oxidant N-acetylcysteine on the responses to DOPAL were also examined. DOPAL concentration-dependently evoked oxidative stress, decreased the mitochondrial membrane potential, activated caspase, and increased early and late apoptosis. In test tubes DOPAL was converted to DOPAL-quinone, quantified by near-infrared fluorescence. Incubation of DOPAL with synuclein oligomerized the proteins, and NAC attenuated both DOPAL-induced generation of reactive oxygen species and alpha-synuclein oligomerization. Preliminarily, from these results we infer that DOPAL toxicity involves oxidative stress and protein modifications, with DOPAL oxidation contributing to the protein modifications. REFERENCES 1. Goldstein DS, Sullivan P, Holmes C, Mash DC, Kopin IJ, Sharabi Y. Determinants of denervation-independent depletion of putamen dopamine in Parkinson's disease and multiple system atrophy. Parkinsonism Relat Disord 2017;35:88-91. 2. Jinsmaa Y, Sullivan P, Sharabi Y, Goldstein DS. DOPAL is transmissible to and oligomerizes alpha-synuclein in human glial cells. Auton Neurosci 2016;194:46-51. 3. Isonaka R, Holmes C, Cook GA, Sullivan P, Sharabi Y, Goldstein DS. Pure autonomic failure without synucleinopathy. Clin Auton Res 2017;27:97-101.